The extreme heterogeneity of human cancers, due to a wide variety of genetic alterations and microenvironmental stresses, presents significant challenges for effective treatments. Robust aerobic glycolysis and significant perfusion defects cause an accumulation of lactic acid, termed lactic acidosis, in many solid human tumors. Evidence is accumulating for an active role of lactic acidosis in affecting tumor phenotypes, treatment responses and clinical outcomes. While lactic acidosis inhibits tumor growth and glycolysis, cancer cells that survive exposure to lactic acidosis for an extended period of time often metastasize and resist radio- and chemotherapeutics. Therefore, specifically targeting cancer cells under lactic acidosis will help reduce treatment resistance and improve clinical outcomes. Although significant efforts have been mead to target cells under hypoxia, relatively little attention has been paid to targeting cells under lactic acidosis. To fill this gap, we propoe a systematic approach to discover strategies to specifically target cells under lactic acidosis by applying the concept of synthetic lethality - genes whose disruptions, while normally tolerated, confer lethality under lactic acidosis. First, we will integrate metabolomic and transcriptional profiling of human cancer cells to identify the metabolic inflexibilities and bioenergetic restrictions imposed by lactic acidosis. Second, we will perform genome-wide, synthetic lethal RNAi screens to identify genes which are essential for survival only under lactic acidosis. Then through both genetic and chemical inhibition, we will evaluate strategies for targeting the identified pathways and genes that are critical for cells under lactic acidosis. In addition, we wil test whether the DNA amplification of the contextually- essential genes confers any survival advantage under lactic acidosis and renders cells uniquely susceptible to their targeting. This proposal presents an innovative and integrative approach to identify novel strategies to eradicate cancer cells under lactic acidosis to achieve a tangible and positive impact on patients' outcomes.